Antibiotic resistance is the ability of a microorganism to withstand the effects of an antibiotic. This resistance develops through genetic mutation and plasmid exchange between microorganisms. Already, antibiotic resistance is having a major impact on medicine that will only increase in the coming years.
One group of the opportunistic microorganisms that gain renewed interest for exhibiting antibiotic resistance are the Enterobacteriaceae. These bacterial species (including for example Klebsiella spp and Escherichia coli) comprise opportunistic pathogens that have i. a. been associated with urinary tract infections, septicaemia, respiratory tract infections and diarrhoea. Resistance of these species to third generation cephalosporins such as oxyimino beta-lactams has been known for 30 years but an exponential increase in resistance has since been recorded. Strains gain their resistance by producing so-called extended-spectrum beta-lactamases (ESBLs), which are Molecular Class A beta-lactamases, capable of inactivating third-generation cephalosporins (ceftazidime, cefotaxime, and cefpodoxime) as well as monobactams (aztreonam). ESBLs are derivatives of common beta-lactamases (e.g. TEM and SHV beta-lactamases) that have undergone one or more amino acid substitutions near the active site of the enzyme, thus increasing their affinity for and hydrolytic activity against third generation cephalosporins and monobactams. Extensive use of newer generation cephalosporins drives the evolution of an increasing range of new ESBLs. ESBLs are encoded by transferable conjugative plasmids that are responsible for the dissemination of resistance to other members of gram negative bacteria.
ESBLs are distinguished into more than 450 types based on their physical properties and are variably inhibited by clavulanate, sulbactam and tazobactam, a property which has been used to detect them in the laboratory. Currently, only phenotypic ESBL detection tests are used in the clinical microbiology laboratory. Molecular (genotypic) tests are under development. A problem with molecular tests is, however, the lack of a 100% correlation between the genotype and the phenotype. Hence, the predictive value of molecular testing for any bacterial phenotype, including ESBL producing bacteria, is limited.
In general, the current phenotypic laboratory tests are sensitive and specific as compared to ESBL genotypic confirmatory tests. All phenotypic ESBL detection tests rely on the same principle: the tests assess variation in the inhibition of bacterial growth in the presence of beta-lactam antibiotics or combinations of beta-lactam antibiotics and beta-lactamase inhibitors. Various manual tests and automated platforms are commercially available for performing these phenotypic tests. The manual tests use disks or strips impregnated with beta-lactam antibiotics or combinations of beta-lactam antibiotics and beta-lactamase inhibitors. The impregnated material is placed on solid media that is pre-inoculated with a bacterial suspension of known density. Following overnight incubation, growth inhibition is determined visually and can be quantified on the basis of the diameter of inhibition zones. The automated systems are also based on measurement of bacterial growth in the presence of panels of beta-lactam antibiotics or combinations of beta-lactam antibiotics and beta-lactamase inhibitors at different concentrations. Results of such systems are obtained after 4 h-18 h.
There is presently a need for means and methods that are capable of diagnosing ESBL producing bacteria more rapidly. There is also a need for an ESBL detection test that can be used in the clinical microbiology laboratory to characterize the ESBL enzymes in terms of enzyme kinetics in order to track evolutionary trends and evaluate and predict the effective dosage in antibiotic therapy. Preferably, such means find wider applicability in characterizing antibiotic resistance in microorganisms in general.